Name: analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions
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Description: Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1. Preparativi per colture cellulari <ol><li> Acquisto commercialmente disponibili scorte di cellule umane. NOTA: Questo protocollo utilizza il carcinoma del colon-retto HCT116 e renali cellule primarie umane prossimali tubulari epiteliali (HRPTEC). </li><li> Fare 500 ml di mezzo per la cultura di HCT116: Modified Eagle Medium di Dulbecco (DMEM) / medio alta di glucosio integrato con 7,5% siero fetale bovino (FBS) e 1% penicillina / streptomicina (P / S). </li><li> Fare 500 ml di mezzo per la cultura di HRPTEC:...

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L&#39;analisi delle proteine ​​di legame della cap in cellule umane esposte a condizioni di ossigeno fisiologiche può consentire l&#39;identificazione di nuovi fattori di inizio della traduzione di ossigeno-regolamentato. L&#39;affinità di questi fattori per tappo di mRNA o di altre proteine cap-associate al 5 &#39;può essere misurata con la forza della loro associazione di m 7 GTP-linked perline agarosio. Un avvertimento di questa tecnica è che misura il potenziale cap-legame delle proteine ​​pos...

Il controllo traslazionale sta emergendo come un passo altrettanto importante regolazione trascrizionale dell&#39;espressione genica, in particolare durante i periodi di stress cellulare 1. Un punto focale di controllo traduzione è al fattore limitante di iniziazione dove le prime fasi della sintesi proteica coinvolgono il legame della eucariotica fattore di iniziazione 4E (eIF4E) al 7-methylguanosine (m 7 GTP) 5 &#39;tappo di mRNA 2 . eIF4E è parte di un complesso di nome trimeric eIF4F che include eIF4A, una elicasi RNA, e eIF4G, una proteina impalcatura necessaria per l&#39;assunzione di altri fattori di traduzione e le 40S del ribosoma 3. In condizioni fisiologiche normali, la stragrande maggioranza degli mRNA vengono tradotti attraverso un meccanismo di cap-dipendente, ma sotto i periodi di stress cellulare circa il 10% di mRNA umani contiene 5 &#39;UTR che potrebbero consentire di traduzione cap-indipendente rito di iniziazione 1,4. traduzione Cap-dipendente è stata storicamente sinonimounità organizzative con eIF4F, tuttavia, le variazioni specifiche di stress di eIF4F sono diventati un argomento di tendenza 5-8. Vari stress cellulari causano attività eIF4E da reprimere attraverso la mammalian target of complesso rapamicina 1 (mTORC1). Questa chinasi diventa compromessa sotto stress, che provoca l&#39;aumento di attività di uno dei suoi obiettivi, la proteina 4E-binding (4E-BP). Non fosforilata 4E-BP si lega ad eIF4E e blocca la sua capacità di interagire con eIF4G causando la repressione della traduzione cap-dipendente 9,10. È interessante notare che, un omologo di eIF4E chiamato eIF4E2 (o 4EHP) ha un&#39;affinità molto più basso per 4E-BP 11, forse permettendogli di eludere la repressione dello stress-mediata. Infatti, inizialmente caratterizzato come un repressore della traduzione a causa della sua mancanza di interazione con eIF4G 12, eIF4E2 avvia la traduzione di centinaia di mRNA che contengono RNA elementi di risposta all&#39;ipossia nella loro 3 &#39;UTR durante lo stress ipossico 6,13. Questo mi attivaziones raggiunto attraverso le interazioni con eIF4G3, RNA-binding protein 4 motivo, e il fattore inducibile dall&#39;ipossia (HIF) 2α a costituire un complesso eIF4F ipossica, o eIF4F H 6,13. Come un repressore in condizioni normali, eIF4E2 si lega con GIGYF2 e ZNF598 14. Questi complessi sono stati, in parte, identificati attraverso Agarose-linked m 7 resine GTP affinità. Questo metodo classico 15 è standard nel campo della traduzione ed è i saggi migliore e più tecnica comunemente usata per isolare complessi cap-binding in pull down e in vitro di legame 16-19. Come le macchine di traduzione cap-dipendente sta emergendo come flessibile ed adattabile con le parti tra loro cambiare 6-8,13, questo metodo è un potente strumento per individuare rapidamente nuove proteine di legame della cap coinvolti nella risposta allo stress. Inoltre, le variazioni di eIF4F potrebbe avere vaste implicazioni come diversi sistemi modello eucarioti sembrano utilizzare un omologo eIF4E2 per le risposte di stress talicome A. thaliana 20, S. pombe 21, D. melanogaster 22, e C. elegans 23. L&#39;evidenza suggerisce che le variazioni di eIF4F non possono essere strettamente limitato a condizioni di stress, ma essere coinvolti nella fisiologia normale 24. La fornitura di ossigeno ai tessuti (alle estremità capillari) o all&#39;interno dei tessuti (misurata tramite microelettrodi) varia dal 2-6% nel cervello 25, 3-12% nei polmoni 26, 3.5-6% nell&#39;intestino 27, 4% in il fegato 28, 7-12% nel rene 29, 4% nel muscolo 30, e 6-7% nel midollo osseo 31. Le cellule e mitocondri contengono meno del 1,3% di ossigeno 32. Questi valori sono molto più vicini a ipossia che l&#39;aria ambiente in cui le cellule sono normalmente coltivate. Questo suggerisce che ciò che è stato precedentemente pensato come processi cellulari specifici per ipossia possono essere rilevanti in un ambiente fisiologico. È interessante notare che, eIF4F e eIF4F H </sup&gt; partecipare attivamente al inizio della traduzione di piscine distinte o classi di mRNA in diverse linee cellulari umane differenti esposti all&#39;ossigeno fisiologica o &quot;physioxia&quot; 24. Low ossigeno spinge anche il corretto sviluppo del feto 33 e le cellule hanno generalmente più alti tassi di proliferazione, durata della vita più lunga, meno danni al DNA e meno risposte allo stress generale in physioxia 34. Pertanto, eIF4F H è probabilmente un fattore chiave per l&#39;espressione dei geni selezionati in condizioni fisiologiche. Qui, forniamo un protocollo per le cellule di coltura in condizioni di ossigeno fisiologiche fissi o in un intervallo di fluttuazione dinamica che rischia più rappresentativa di microambienti tissutali. Un vantaggio di questo metodo è che le cellule sono lisate nella workstation ipossia. E spesso non è chiaro come la transizione dalla coltura cellulare ipossica a lisi cellulare avviene in altri protocolli. Le cellule vengono spesso rimossi prima da un piccolo incubatore ipossia esserefore lisi, ma questa esposizione all&#39;ossigeno potrebbe influenzare percorsi biochimici come risposta cellulare a ossigeno è rapida (uno o due min) 35. Alcune proteine di legame della cap richiedono interazioni con una seconda base o possono idrolizzare il GTP m 7, quindi alcuni interattori capitalizzazione possono perdere nel processo di purificazione. Agarosio-linked per analoghi cap enzimaticamente resistenti può essere sostituito in questo protocollo. Esplorare l&#39;attività e la composizione di eIF4F H e altre varianti del eIF4F attraverso il metodo descritto qui farà luce sui complessi macchinari di espressione genica che le cellule utilizzano in condizioni fisiologiche o risposte allo stress.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:380px;">&#947;-aminophenyl-m7GTP agarose C10-linked beads</td>
			<td style="width:151px;">Jena Bioscience</td>
			<td style="width:165px;">AC-1555</td>
			<td style="width:205px;">Agarose-linked m7GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dish</td>
			<td>Corning</td>
			<td>877222</td>
			<td>10-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm culture dish</td>
			<td>Thermofisher</td>
			<td>130183</td>
			<td>15-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AEBSF Hydrochloride</td>
			<td>ACROS Organics</td>
			<td>A0356829</td>
			<td>AEBSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose Beads</td>
			<td>Jena Bioscience&#160;</td>
			<td>AC-0015</td>
			<td>Agarose bead control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Fisher</td>
			<td>BP112-25</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL Centrifuge Tubes</td>
			<td>FroggaBio</td>
			<td>1210-00S</td>
			<td>Used to centrifuge small volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
			<td>Used in culturing primary cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined trypsin inhibitor</td>
			<td>Fisher</td>
			<td>R007100</td>
			<td>DTI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial cell medium (complete kit)</td>
			<td>ScienCell</td>
			<td>4101</td>
			<td>Includes serum and growth factor supplements)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Guanosine 5&#39;-triphosphate, 1 mL</td>
			<td>Jena Bioscience</td>
			<td>272076-0251M</td>
			<td>GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 colorectal carcinoma</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>Human cancer cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human renal proximal tubular epithelial cells</td>
			<td>ATCC</td>
			<td>PCS-400-010</td>
			<td>HRPTEC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone DMEM/High Glucose</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td>Standard media for human cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Penicillin-Streptomycin solution</td>
			<td>GE Life Sciences</td>
			<td>SV30010</td>
			<td>Antibiotic component of DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H35 HypOxystation</td>
			<td>Hypoxygen</td>
			<td>N/A</td>
			<td>Hypoxia workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Igepal CA-630</td>
			<td>MP Biomedicals</td>
			<td>2198596</td>
			<td>Detergent component of lysis buffer</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Monopotassium phosphate</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>KCl</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td>MgCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
			<td>NaF (phosphatase inhibitor component of lysis buffer)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Disodium phosphate</td>
			<td>Fisher</td>
			<td>5369-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
			<td>58715</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100</td>
			<td>SDS</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>56508</td>
			<td>Na3VO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td>Component of buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>2500-067</td>
			<td>Trypsin used to detach adherent cells</td>
		</tr>
	</tbody>
</table>

analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Analisi di Capacità Cap-binding in risposta ad ossigeno di eIF4E e eIF4E2 in un m 7 GTP Affinity Colonna Le figure 1 e 2 rappresentano le macchie occidentali del tipico m 7 GTP affinità purificazione delle due principali proteine di legame della cap in risposta alle fluttuazioni di ossigeno in due linee cellulari umane: umane renale prossimale cellule primarie tubulari epi...

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Analisi delle proteine ​​di legame della cap in cellule umane esposte a condizioni fisiologiche di ossigeno

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

The overall goal of this experiment is to capture cap-binding proteins in conditions of low oxygen. This method utilizes an agarose linked seven methyl-guanosine cap analog to investigate cap-binding factors and their interacting partners. This method will help answer key questions in the field of protein synthesis such as identifying novel oxygen regulated factors involved in cap dependent translation.The main advantage of this technique is that cells are kept in a hypoxia workstation up until the point of lysis. The implications of this technique extend towards cancer therapy because cancer cells within a tumor are exposed to very low levels of oxygen and translational control plays a key role in tumor progression. We first had the idea for this method when we read a recent publication from Corona's colleagues reporting that tissue oxygenation is actually closer to what we call hypoxia rather than the normaxia that cells are routinely cultured in.Begin this procedure will cell culture as described in the text protocol. After counting the cells using a hemocytometer feed 150 millimeter cultured dishes containing 15 milliliters of complete medium to obtain a cell density of 2500 to 5000 cells per square centimeter. Use two 150 millimeter dishes for each cap-binding assay.Place the seeded culture dishes in the incubator at 37 degrees celsius and a five percent carbon dioxide atmosphere. After the cells have incubated at least 24 hours place the cells into a hypoxia workstation for the desired time. Then, set the workstation to the appropriate physioxia.Pre-warm one X phosphate buffered saline, or PBS, and trypsin in a 37 degree celsius water bath for 30 minutes. Aliquot 980 microliters of lysis buffer into a 1.5 microliter centrifuge tube for each cap-binding assay being performed. 10 microliters of 100X protease inhibitor cocktail and 10 microliters of 4-benzenesulfonyl fluoride hydrocloride to the 980 microliters of lysis buffer on ice.Discard the media from each 150 millimeter dish in a waste container within the hypoxia workstation. Then, wash the cells with three to four milliliters of warm one X PBS and discard the liquid. Add one milliliter of warm 05 percent trypsin EDTA to each dish and let the dishes sit for two minutes or until the cells are no longer adhered to the plate.Using a pipette transfer the cells of one plate to a 1.5 milliliter micro-centrifuge tube. Repeat as needed so that each plate of cells is transferred to a separate 1.5 milliliter micro-centrifuge tube. Centrifuge the micro-centrifuge tubes at 6000 times g for 90 seconds to pellet the cells.Following centrifugation aspirate the trypsin using a pipette without disturbing the pellet. Wash the cells with warm one X PBS by gently pipetting 200 microliters of PBS into the tube just above the pellet. Then aspirate the PBS without disturbing the pellet.Next, pipette 500 microliters from the one milliliter prepared lysis buffer solution onto the first cellular pellet. Re-suspend the pellet entirely by pipetting up and down. Combine the re-suspended cells with the second cell pellet and re-suspend the second pellet by pipetting up and down.Repeat this process until all pellets have been combined and re-suspended for each sample. Next, combine the lysate with the remaining 500 microliters of lysis buffer in a 1.5 liter micro-centrifuge tube. Remove the samples from the hypoxia workstation.Lyse the cells using gentle agitation by rotating the samples at four degrees celsisus for 1.5 to two hours. Following incubation, centrifuge the lysed cells at 1200 times g for 15 minutes at four degrees celsius to remove cellular debris. Transfer the lysate to a new 1.5 milliliter micro-centrifuge tube and discard the pellet.Reserve a portion of the supernatant to be used as whole cell lysate input control for western blot analysis. To prepare for the cap-binding assay use scissors to remove the tip of the pipette to facilitate collection of the bead slurry. For each sample transfer 50 microliters of the blank agarose bead control slurry and 50 microliters of the m7GTP agarose C10-linked beads slurry to separate 1.5 milliliter micro-centrifuge tubes.Then, pellet the beads by centrifuging the slurry at 500 times g for 30 seconds. Remove the supernatant carefully and re-suspend the beads in 500 microliters of TBS. After repeating these steps pellet the beads at 500 times g for 30 seconds and remove the supernatant.Next, transfer the supernatant containing the lysate to the 1.5 milliliter micro-centrifuge tube containing the blank agarose beads. Incubate for 10 minutes at four degrees celsius with gentle agitation. After 10 minutes, pellet the blank agarose beads by centrifuging at 500 times g for 30 seconds.Following centrifugation transfer the lysate that doesn't bind to the beads to the 1.5 milliliter micro-centrifuge tube containing the m7GTP agarose C10-linked beads. Wash the blank agarose beads by re-suspending the beads in 500 microliters of lysis buffer, pelleting the beads by centrifuging at 500 time g for 30 seconds, and discarding the supernatant. Repeat this four times for a total of five washes.Re-suspend the blank agarose beads in one X SDS-PAGE sample buffer. And boil the beads for 90 seconds at 95 degrees celsius. Store the sample at 20 degrees celsius for future western blot analysis to observe weather the protein of interest binds non-specifically to the bead.Incubate the lysate with the m7GTP agarose C10-linked beads with gentle agitation for one hour at four degrees celsuis to capture cap-binding proteins. Following agitation, pellet the m7GTP agarose C10-linked beads by centrifuging at 500 times g for 30 seconds. After discarding the supernatant, wash the m7GTP agarose C10-linked beads as before.Re-suspend the beads in 600 microliters of lysis buffer and add GTP to a final concentration of one millimolar. Incubate the m7GTP agarose C10-linked beads plus one millimolar GTP with gentle agitation for one hour at four degrees celsius. This will dissociate proteins that non-specifically interact with m7GTP.Pellet the m7GTP agarose C10-linked beads by centrifuging at 500 time g for 30 seconds. Transfer the supernatant to a new 1.5 milliliter micro-centrifuge tube, and add 200 microliters of four X SDS-PAGE sample buffer. After washing the beads as before, re-suspend the beads in one X SDS-PAGE sample buffer and boil at 95 degrees celsius for 90 seconds.Shown here are representative western blots of a typical m7GTP affinity purification of two major cap-binding proteins in response to oxygen fluctuations in primary human renal proximal tubule epithelial cells. The cell lysates were exposed to eight percent, five percent, three percent, or one percent oxygen for 24 hours. The lanes include 10 percent of the whole cell lysate as the input, a GTP washed to measure the specificity for m7GTP, and the proteins bound to m7GTP beads after the GTP wash.Data from at least three independent experiments were quantified by Image J and expressed as relative density units relative to 10 percent input of the whole cell lysate. Results indicate that human cells cultured under physiological oxygen utilize two cap-binding proteins to recruit distinct mRNAs for translation. After watching this video you should have a good understanding of how to capture cap-binding proteins in low oxygen conditions.While attempting this procedure it's important to remember to keep cells in the hypoxia workstation up until the point of lysis, as oxygen can rapidly alter the biochemical properties of these translation initiation factors. Following this procedure other methods like polysome fractionation can be performed to answer additional questions such as which cap-binding proteins associate with active translation under conditions of low oxygen.

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